The invention relates to methods for implanting cells onto prosthetic materials, as well as methods for generating such implantable cells.
Despite prevention efforts, atherosclerotic disease remains a major cause of morbidity and mortality. Treatments for atherosclerotic disease range from medical management to interventional therapies, such as angioplasty, atherectomy, and bypass grafting. Bypass grafting with synthetic grafts has received much attention and has been used to treat many patients. Unfortunately, small caliber vascular grafts (i.e., grafts with inner diameters of less than 6 mm) generally have high failure rates, due largely to the thrombogenicity of the grafts. Thrombus deposits can form on the inner walls of the grafts, resulting in occlusions. In addition, intimal hyperplasia can occur, further contributing to the failure of small caliber grafts.
Several strategies for improving the success rates of these grafts have been developed. One such strategy is to increase the rate at which the graft becomes endothelialized, as endothelial cells have natural anti-thrombogenic properties that contribute to long-term graft patency. Moreover, it is believed that the presence of endothelial cells inhibits the development of neointimal hyperplasia at anastomotic regions. Endothelialization, which involves the migration of endothelial cells from adjacent tissue onto the luminal surface, can occur spontaneously when a graft is placed in a recipient. Unfortunately, endothelialization occurs to only a limited degree when prosthetic grafts are placed in human recipients, and the limited endothelialization that does occur takes place slowly.
To promote the rapid formation of an endothelial lining, endothelial cells can be seeded or sodded onto a graft before the graft is placed in the recipient. When the graft is placed in the recipient and exposed to physiologic blood flow, however, these cells are often washed away.
In general, the invention features a method for implanting cells onto a prosthesis; the method includes the steps of: (a) providing a prosthesis including a porous tube, where at least 25% of the pores on the inner surface of the tube have diameters of more than about 40 xcexcm, at least 25% of the pores on the outer surface of the tube have diameters of less than about 30 xcexcm, and the tube includes a substantially continuous layer of a biocompatible material; (b) contacting the prosthesis with a suspension of cells; and (c) providing a pressure differential between the inner surface and the outer surface, whereby the cells are retained in the pores of the inner surface. An example of a prosthesis that can be used is a vascular graft. The invention also features a sodded prosthesis formed by this method.
Preferably, at least 50% of the pores on the inner surface of the tube have diameters of more than about 40 xcexcm, and at least 50% of the pores on the outer surface of the tube have diameters of less than about 30 xcexcm. More preferably, at least 70%, or at least 90%, of the pores on the inner surface of the tube have diameters of more than about 40 xcexcm, and at least 70%, or at least 90%, of the pores on the outer surface of the tube have diameters of less than about 30 xcexcm.
In other preferred methods, at least 25% of the pores on the inner surface have diameters of more than about 50 xcexcm, and more preferably have diameters of about 60 xcexcm; in addition, at least 25% of the pores on the outer surface have diameters of less than about 20 xcexcm, and more preferably have diameters of less than about 15 xcexcm. More preferably, at least 50%, 70%, or 90% of the pores on the inner surface have diameters of more than about 50 xcexcm, and more preferably have diameters of about 60 xcexcm; in addition, at least 50%, 70%, or 90% of the pores on the outer surface have diameters of less than about 20 xcexcm, and more preferably have diameters of less than about 15 xcexcm.
Preferably, the pores on the inner surface and the pores on the outer surface are connected by gradually tapered openings.
In a related aspect, the invention features a method for implanting cells onto a prosthesis; the method includes the steps of: (a) providing a prosthesis including a porous tube, where the diameters of at least 25% of the pores on the inner surface of the tube are larger than the diameter of a human cell, such that human cells fit within the pores, the diameters of at least 25% of the pores on the outer surface of the tube are smaller than the diameter of a human cell, such that cells do not pass through the pores, and the tube includes a substantially continuous layer of a biocompatible material; (b) contacting the prosthesis with a suspension of cells; and (c) providing a pressure differential between the inner surface and the outer surface, whereby the cells are retained in the pores of the inner surface.
Preferably, the pores on the inner surface and the pores on the outer surface are connected by gradually tapered openings. In addition, the diameters of at least 50%, 70%, or 90% of the pores on the inner surface of the tube are preferably larger than the diameter of a human cell, and the diameters of at least 50%, 70%, or 90% of the pores on the outer surface of the tube are preferably smaller than the diameter of a human cell. The invention also features a sodded prosthesis formed by this method.
In another related aspect, the invention features a sodded vascular graft including a porous tube, where at least 25% of the pores on the inner surface of the tube have diameters of more than about 40 xcexcm, at least 25% of the pores on the outer surface of the tube have diameters of less than about 30 xcexcm, and the tube includes a substantially continuous layer of a biocompatible material; the graft has cells embedded in the pores of the inner surface. Preferably, the pores on the inner surface and the pores on the outer surface are connected by gradually tapered openings. In preferred grafts, at least 50%, 70%, or 90% of the pores on the inner surface of the tube have diameters of more than about 40 xcexcm, and at least 50%, 70%, or 90% of the pores on the outer surface of the tube have diameters of less than about 30 xcexcm.
In yet another related aspect, the invention features a sodded vascular graft including a porous tube, where the diameters of at least 25% of the pores on the inner surface of the tube are larger than the diameter of a human cell, the diameters of at least 25% of the pores on the outer surface of the tube are smaller than the diameter of a human cell, such that cells do not pass through the pores, and the tube includes a substantially continuous layer of a biocompatible material; the graft has cells embedded in the pores of the inner surface. Preferably, the pores on the inner surface and the pores on the outer surface are connected by gradually tapered openings. In preferred grafts, the diameters of at least 50%, 70%, or 90% of the pores on the inner surface of the tube are larger than the diameter of a human cell, and the diameters of at least 50%, 70%, or 90% of the pores on the outer surface of the tube are smaller than the diameter of a human cell.
In a final aspect, the invention features a method for obtaining an endothelial cell culture from a blood sample, the method involving: (a) obtaining a sample of mononuclear cells from a blood sample; and (b) culturing the sample of mononuclear cells, without further cell separation, on a cell adhesive polymer-coated solid support in the presence of endothelial growth factors.
In preferred embodiments, the blood sample is from a mammal (for example, a human) and the product of step (b) is an autologous endothelial cell sample; the blood sample is a peripheral blood sample; the mononuclear cells are obtained from the blood sample by centrifugation; the cell adhesive polymer is fibronectin; the solid support is a tissue culture plate; the endothelial growth factors include VEGF, bFGF, IGF, or any combination thereof; and the endothelial cell culture includes at least 90% endothelial cells or progenitors thereof.
As used herein, by xe2x80x9csubstantially continuousxe2x80x9d is meant that a material, such as the material forming the walls of a prosthesis, consists essentially of a single layer having approximately constant, or gradually changing, physical characteristics.
By xe2x80x9cgradually taperedxe2x80x9d is meant that a dimension, such as pore size, changes in even, continuous gradations, rather than in discrete, sudden steps.
By xe2x80x9cembeddedxe2x80x9d is meant that at least a portion of an object, such as a cell, infiltrates and is substantially enclosed by a surrounding structure or medium.
By xe2x80x9cretainedxe2x80x9d is meant held in place. For a vascular graft, at least 20%, more preferably at least 50%, and most preferably at least 90%, of the cells remain in place in the graft under physiological blood flow conditions after a period of 14 days.
By an xe2x80x9cendothelial cell culturexe2x80x9d is meant a population of cells having at least 50%, preferably, at least 75%, more preferably, at least 80%, and, most preferably, at least 90% endothelial cells or progenitors thereof.
By a xe2x80x9cblood samplexe2x80x9d is meant any biological sample composed primarily of hematopoietic cells including, without limitation, a peripheral blood sample, bone marrow blood sample, or umbilical cord blood sample.
By a xe2x80x9csample of mononuclear cellsxe2x80x9d is meant a population of cells having at least 40%, preferably, at least 60%, more preferably, at least 75%, and, most preferably, at least 90% mononuclear cells.
By xe2x80x9ccell separationxe2x80x9d is meant any technique which physically subdivides a cell population including, without limitation, column chromatography, immunomagnetic separation, immunoprecipitation, and cell sorting techniques (for example, fluorescence-activated cell sorting).
By a xe2x80x9ccell adhesive polymerxe2x80x9d is meant any polymer which provides a substrate for endothelial cell attachment including, without limitation, fibronectin, vitronectin, laminin, keratin, gelatin, and collagen, with fibronectin being preferred.
By an xe2x80x9cendothelial growth factorxe2x80x9d is meant any protein which stimulates growth or differentiation of endothelial cells or their progenitors, including, without limitation, vascular endothelial cell growth factor (VEGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF).
By a xe2x80x9csolid supportxe2x80x9d is meant any solid surface which can support cell growth or differentiation including, without limitation, a tissue culture plate or well, bead, slide, column, bottle, or other vessel.
The cell implantation methods of the invention offer several advantages. For example, because the pores on the inner surfaces of the prostheses are relatively large, cells are retained on the inner surface, rather than being washed away by blood flow. The smaller pores on the outer surface of the prostheses prevent the cells from passing through the walls of the prosthesis. The smaller pores also help to maintain the structural integrity of the prosthesis and to reduce the propensity for bleeding that can occur, for example, in vascular grafts with uniformly large pores. These cell implantation methods therefore enhance cell retention without compromising the safety of the prostheses.
Furthermore, using the implantation methods of the invention, cells can be retained without the addition of adhesive substances such as the matrix proteins laminin and fibronectin, which are sometimes applied to the surface of prosthetic materials to enhance cell retention. As the presence of these proteins can lead to increased thrombogenicity, the ability to retain cells without them may increase the chances for a successful graft.
With respect to the methods for culturing endothelial cells to be implanted, the present invention again provides significant advantages. In particular, because the method described herein utilizes a standard blood sample as a source of endothelial cells, it avoids the need to obtain such cells by surgical removal of autologous veins or adipose tissue, thereby reducing patient trauma. Moreover, the present culture method provides a means for expanding an endothelial cell culture which does not require a physical cell separation step to remove unrelated, hematopoietic cells from the mononuclear blood fraction. The ability to dispense with this step makes the technique extremely rapid and quite cost-effective.